Objective To develop a multiplex touchdown PCR for simultaneous detection of extended-spectrum β-lactamases (ESBLs )-producing Enterobacteriaceae and methicillin-resistant Staphylococcus aureus (MRSA ). Methods Blood culture positive specimens from 102 hospitalized patients were collected between March 2013 and September 2014,four pairs of specific primers were designed based on SHV,TEM,and OXA genes of ESBLs-pro-ducing Enterobacteriaceae and MecA gene of MRSA,drug-resistant genes were amplified with single touchdown PCR and multiplex touchdown PCR, the results were compared with Kirby-Bauer disk diffusion method. Results Each single PCR amplified a specific band,four drug-resistant genes were also detected by multiplex touchdown PCR;the lower detection limits of multiplex touchdown PCR for DNA of MecA,SHV,TEM,and OXA were 4.37 ng,2.19 ng,4.53 ng,and 3.59 ng,respectively.Compared with Kirby-Bauer disk diffusion method, the overall sensitivity and specificity of multiplex touchdown PCR were 100.00% and 88.24% respectively,for ES-BLs were 100.00% and 87.23% respectively,for MRSA were both 100.00%.Conclusion A higher sensitivity and specificity multiple touchdown PCR assay has been developed,and it can be used in the rapid diagnosis and epidemi-ology investigation of bloodstream infection caused by ESBLs-producing Enterobacteriaceae and MRSA,and is help-ful for guiding antimicrobial use in clinic.